1. Field of the Invention
The present invention relates generally to the fields of immunology, molecular biology, and cellular biology. More particularly, it relates to quantitative multiplexed cellular analysis using flow cytometry, microscopy, and/or fluorimetry.
2. Description of Related Art
Multiplexed target labeling and analysis are principal strategies applied in molecular biology research. In general, surface or intracellular antigens indicative of cell status are detected by multiplexed labeling with targeting reagents (e.g., antibodies), followed by visualization of the targeting reagents by specific labeling probes (e.g., fluorophores). In some instances, targets in solution are analyzed using a similar approach. The sample is analyzed by flow cytometry, microcopy, fluorimetry, or other instrumentation equipped to measure labeling probe signal.
Multiplexed targeting assays have been facilitated in the last several decades by an increasing variety of commercially available antibodies biochemically conjugated to labeling reagents. Fluorescent reagents are the most common type of label used in the laboratory, although other labels may be utilized for specific applications (enzymes, radioisotopes, heavy metals, etc). Despite the growing availability of directly-labeled targeting reagents, the majority of reagents are only available conjugated to a limited number of labels, often in the same standard fluorophore such as FITC. This is particularly true of reagents targeting novel or niche markers.
A variety of parameters must be considered in order to determine an optimal multiplexed detection strategy, including cell type(s), target densities, labeling reagent characteristics, and instrument specifications. Limitations placed on label-target choice by commercial availability, coupled with reliance on qualitative analysis parameters, can cause variation in results and subsequent interpretation of data across experiments, researchers, and laboratories.
The prevalence of qualitative, rather than quantitative, analysis in many immunoassays is a result of several factors. Qualitative analysis is almost universally practiced with flow cytometric and microscopy assays, due to the nature of instrumentation, which are configured to provide a measure of adjustable, relative intensity, rather than units of absolute intensity. While some quantitative technologies exist, such as dyed fluorescent microspheres, at present these technologies require an additional investment of cost and preparation time that may deter many researchers, and even when utilized may not produce reliable and accurate quantitative measures. Although immunoanalysis procedures are, by and large, executed by researchers with considerable experience and expertise, there is no question that a streamlined method of accurate, quantitative analysis would represent a significant asset to the field—notably, for flow cytometric applications which are particularly subject to variation and error incurred by the qualitative approach.
As existing technologies are often time-consuming, cumbersome, and inaccurate, it is understandable that the quantitative analysis endeavor is not usually pursued by the research laboratory.